Label-free methods for isolation and analysis of nucleic acids on solid phase device

ABSTRACT

Methods and system for the isolation and/or analysis of nucleic acids on a solid phase device comprising (i) incubating a nucleic acid sample with Dimethyl adipimidate (DMA) on said solid phase under conditions that allow formation of a complex of the nucleic acid with the DMA; contacting the complex of (i) with said surface; and isolating and/or analyzing the nucleic acid of the complex.

CROSS-REFERENCE TO RELATED APPLICATIONS

This patent application is a divisional application of U.S. patentapplication Ser. No. 14/652,099, filed Jun. 12, 2015, entitled“LABEL-FREE METHODS FOR ISOLATION AND ANALYSIS OF NUCLEIC ACIDS ON SOLIDPHASE DEVICE,” which is a U.S. National Phase Application under 35U.S.C. § 371 of International Application No. PCT/SG2013/000533, filedon 13 Dec. 2013, entitled LABEL-FREE METHODS FOR ISOLATION AND ANALYSISOF NUCLEIC ACIDS ON SOLID PHASE DEVICE, which claims the benefit ofpriority of Singapore patent application No. 201209173-2, filed on 13Dec. 2012.

FIELD

Methods of isolating nucleic acids preferably on a solid phase device.

BACKGROUND

Nucleic acid is an important analysis tool when identifying a diseasestate. DNA biomarkers (e.g. single nucleotide polymorphism (SNP),mutation, and DNA methylation) offer important clues to help researcherslook for the causes of cancer and provide great opportunities todiagnose and monitor disease status during the early stages of diseases,as well as for prognosis and surveillance. Because of the extremely lowphysiological concentration of DNA compared to other components such asproteins (i.e. tens of nanograms of DNA versus tens of micrograms ofprotein in a microliter of whole blood), efficient extraction andpre-concentration of DNA from clinical samples is critical for thesubsequent downstream processes such as amplification and detection.When it comes to methylated DNA this problem is magnified.

DNA methylation plays a crucial role in the regulation of geneexpression and chromatin organization within normal eukaryotic cells.DNA methylation occurs by covalent addition of a methyl group at the 5carbon of the cytosine ring, resulting in 5-methylcytosine. These methylgroups project into the major groove of DNA and effectively inhibittranscription. In mammalian DNA, 5-methylcytosine is found inapproximately 4% of genomic DNA, primarily at cytosine-guanosinedinucleotides (CpGs). Such CpG sites occur at lower than expectedfrequencies throughout the human genome but are found more frequently atsmall stretches of DNA called CpG islands. These islands are typicallyfound in or near promoter regions of genes, where transcription isinitiated. In contrast to the bulk of genomic DNA, in which most CpGsites are heavily methylated, CpG islands in germ-line tissue andpromoters of normal somatic cells remain unmethylated, allowing geneexpression to occur. DNA methylation is mediated by a family of highlyrelated DNA methyltransferase enzymes (DNMT), which transfer a methylgroup from S-adenosyl-L_methionine to cytosines in CpG dinucleotides.The methyl-cytosines established by the DNMTs serve as binding sites forthe methyl-CpG binding domain (MBD) proteins MeCP2, MBD (S. B. Baylin,DNA methylation and gene silencing in cancer. Nature Clin. Prac. Oncol.2 (2005) 4-11; M. T. McCabe, et al., Cancer DNA methylation: Molecularmechanisms and clinical implications. Clin. Cancer Res. 15 (2009)3927-3937; M. Wielscher, et al. Methyl-binding domain protein-based DNAisolation from human blood serum combines DNA analyses andserum-autoantibody testing. BMC Clin. Pathol. 11 (2011) 11-20; and B. R.Cipriany, et al. Real-time analysis and selection of methylated DNA byfluorescence-activated single molecule sorting in a nanofluidic channel.Proc. Nat. Acad. Sci USA. 109 (2012) 8477-8482. Through interactionswith histone deacetylases, histone methyltransferases, and ATP-dependentchromatin remodeling enzymes, the MBDs translate methylated DNA into acompacted chromatin environment that is repressive for transcription.Especially, MBD is the methyl CpG binding domain of the MeCP2 protein,which binds symmetrically methylated CpGs in any sequence context, andis involved in mediating methylation dependent transcriptionalrepression. Although there is a strong evidence that MeCP2 bindsexclusively methylated DNA fragments in vivo, a DNAmethylation-independent binding activity of MeCP2 in vitro was alsodescribed in concordant literature, which makes it suitable for generalin vitro DNA analysis [S. B. Baylin; McCabe, et al.; M. Wielscher, etal.; and B. R. Cipriany, et al.].

DNA methylation causes silencing of expression of tumor suppressor genesin human cancers. An ever increasing body of work within the field ofepigenomics is strengthening the linkage between the hypermethylation ofkey nucleotide sequences and the advent of many different cancers. DNAmethylation patterns in human cancer cells are considerably distorted.Typically, cancer cells exhibit hypomethylation of intergenic regionsthat normally comprise the majority of a cell's methyl-cytosine content.Consequently, transposable elements may become active and contribute tothe genomic instability observed in cancer cells. Simultaneously, cancercells exhibit hypermethylation within the promoter regions of many CpGisland-associated tumor suppressor genes. As a result, these regulatorygenes are transcriptionally silenced resulting in a loss of function.Thus, through the effects of both hypo- and hyper-methylation, DNAmethylation significantly affects the genomic landscape of cancer cells,potentially to an even greater extent than coding region mutations,which are relatively rare [S. B. Baylin; McCabe, et al.; M. Wielscher,et al.; and B. R. Cipriany, et al]. DNA methylation is of greatimportant for cancer research and clinics, since it enables earliercancer diagnosis prior to the point of metastasis. An example is RARβ, athyroid-steroid hormone receptor that controls the growth of many celltypes by regulating gene expression. Methylation of RARβ has beenreported in breast, lung, and bladder cancers.

The recent development of several genome-scale methylation screeningtechnologies has considerably expanded our understanding of DNAmethylation patterns, both in normal and cancerous cells. Particularly,MSP (methylation-specific PCR), which can rapidly assess the methylationstatus of virtually any group of CpG sites within a CpG island. Thisassay entails initial modification of DNA by sodium bisulfite,converting all unmethylated, but not methylated, cytosines to uracil,and subsequent amplification with primers specific for methylated versusunmethylated DNA. MSP requires only small quantities of DNA, issensitive to 0.1% methylated alleles of a given CpG island locus. Thechemical modification of cytosine to uracil by bisulfite treatment hasprovided another method for the study of DNA methylation that avoids theuse of restriction enzymes. However, these methods are technicallyrather difficult and labor-intensive, and, without cloning of theamplified products, the technique is less sensitive than Southernanalysis, requiring—25% of the alleles to be methylated for detection.Therefore, the isolation of the methylated DNA from human genomic DNA isan important step for improving of DNA methylation analysis in cancer,but that is still challenging [J. G. Herman, J. R. et al.,Methylation-specific PCR: a novel PCR assay for methylation status ofCpG islands. Proc. Nat. Acad. Sci USA. 93 (1996) 9821-9826; S. Pan, etal., Double recognition of oligonucleotide and protein in the detectionof DNA methylation with surface Plasmon resonance biosensors. Biosens.Bioelectron. 26 (2010) 850-853; and J. D. Suter, et al., Label-free DNAmethylation analysis using opto-fluidic ring resonators. Biosens.Bioelectron. 26 (2010) 1016-102].

In solution phase methods, for the isolation of the methylated DNA fromgenomic DNA, up to now, recombinant MBD protein, which is available uponoverexpression of the cloned His-tagged protein in E. coli, has beenpredominantly used for DNA methylation analyses. The MBD protein hasbeen preferably applied being immobilized in an affinity chromatographylike manner with NaCl gradient elution steps to isolate methylated DNAfor PCR and gel analyses in solution phase. According to commercializedprotocol from companies, the MBD protein attached to Ni-Sepharose orMagnetic beads for affinity based DNA purification that enables thesimultaneous analyses of the methylated DNA. MBD isolated the DNA hasbeen found particularly suitable for DNA methylation analyses (FIG. 1,Black_line).

However, the previous studies for the detection of DNA methylation basedlabel-free biosensor without bisulfite modification have been so fardemonstrated only with synthetic oligonucleotides. The direct detectionof native methylated DNAs in genomic DNA in bodily fluids such as blood,urine, or saliva would be difficult due to their extremely lowconcentration. The number of a specific gene in total DNA is extremelylow. For instance, Su et al. have reported that about 2 copies ofmutated tumor Kristin-ras DNA can be found in 50-200 μL of urine orblood samples of cancer patients [Y. H. Su, et al., Block, Detection ofmutated K-ras DNA in urine, plasma, serum of patients with colorectalcarcinoma or adenomatous polyps, Annals of the New York Academy ofSciences 1137 (2008) 197-206]. The sensitivity of the reportedlabel-free biosensors is not good enough to detect such a lowconcentration of native DNA biomarkers. Therefore, these label-freetechniques are inadequate to be used as in vitro diagnostic (IVD) devicewithout amplification of target DNAs.

It has been recently reported that highly sensitive silicon-basedmicroring resonators were used to detect biomolecules (e.g., protein,methylated DNA, nucleic acids) by monitoring a shift in the resonantwavelength. Optical refractive index (RI) sensors are extensivelyinvestigated for a number of applications and play a prominent role inbiochemical analysis. Among the existing biochemical RI sensors, thosebased on integrated optical waveguides are of great interest because oftheir high sensitivity, small size, and high scale integration.Recently, RI sensors based on slot waveguide have attracted significantinterest due to slot waveguide's remarkable property to provide highoptical intensity in a sub-wavelength-size low refractive index region(slot region) sandwiched between two high refractive index strips. Usingthe slot as sensing region, larger light-analyte interaction, and hencehigher sensitivity, can be obtained as compared to a conventionalwaveguide. The sensing light is concentrated close to the surface by anevanescent field undergoing exponential decay with a characteristicdecay length of up to few hundred nanometers. Thus, the refractive indexis affected by the binding of the analyte with immobilized capturingligand within the decay length. Silicon microring resonators arerefractive-index-based optical sensors that provide highly sensitive,label-free, real-time multiplexed detection of biomolecules near thesensor surface. Furthermore, the devices are fabricated by usingstandard CMOS technology, which ensures low cost and scale=upcapability. The methods require time consuming steps of immobilizingprobes on the surface of the device by highly skilled personnel.

The object of the invention is to ameliorate at least some of the abovementioned difficulties.

SUMMARY

Accordingly a first aspect of the invention includes a method for theisolation and/or analysis of nucleic acid molecules on a solid phasedevice comprising

-   -   (i) incubating a nucleic acid sample with Dimethyl adipimidate        (DMA) under conditions that allow formation of a complex of the        nucleic acid with the DMA;    -   (ii) contacting the complex of (i) with a surface of the solid        phase device; and    -   (iii) isolating and/or analyzing the nucleic acid of the        complex.

Another aspect of the invention includes a system for isolating anucleic acid molecule of interest in a nucleic acid sample comprising:

-   -   (i) a Dimethyl adipimidate (DMA) compound capable of directly        binding the nucleic acid molecule; and    -   (ii) a solid surface for the interaction of the nucleic acid and        the DMA.

Other aspects of the invention would be apparent to a person skilled inthe art with reference to the following drawings and description ofvarious non-limiting embodiments.

BRIEF DESCRIPTION OF THE DRAWINGS

The drawings are not necessarily drawn to scale, emphasis insteadgenerally being placed upon illustrating the principles of variousembodiments. In the following description, various embodiments of theinvention are described with reference to the following exemplarydrawings.

FIG. 1. Workflow for isolation and analysis of the DNA using solutionphase versus solid phase methods.

FIG. 2. Complex with DMA and the methylated DNA on solid phase device(#1). Complex with MBD protein and the methylated DNA on solid phasedevice (#2).

FIG. 3. Experimental result of the isolation (A) and analysis (B) of themethylated DNA on solid phase device.

FIG. 4. Experimental result of the isolation with DMA in comparison toadding DNA alone or DMA alone (A). The wavelength shift of themethylated DNA-DMA complex in 2 cancer cell lines (B).

DETAILED DESCRIPTION

Surprisingly DMA (dimethyl adipimidate) can directly bind to nucleicacid without any cross-linked protein intermediary. This directinteraction of DMA and nucleic acid can be used in a label-free methodfor isolation and analysis of nucleic acid or methylated DNA preferablyon solid phase device including silicon, glass, polymer film, plastics,or any suitable solid phase device. The method would be very useful forDNA detection in clinical applications such as human cancer. Directbinding of nucleic acid and DMA provides a simple and cost effectivechemical means of capturing and measuring nucleic acids includingmethylated DNA.

Accordingly a first aspect of the invention includes a method for theisolation and/or analysis of nucleic acid molecules on a solid phasedevice comprising

-   -   (i) incubating a nucleic acid sample with Dimethyl adipimidate        (DMA) under conditions that allow formation of a complex of the        nucleic acid with the DMA;    -   (ii) contacting the complex of (i) with a surface of the solid        phase device; and    -   (iii) isolating and/or analyzing the nucleic add of the complex.

The method allows the isolation and analysis of the nucleic acid to beperformed in a real-time manner.

The term “nucleic acid” as used herein refers to an isolated nucleicacid molecule. The nucleic acid may be DNA. RNA, DNA:RNA hybrids, PNAand the like, but preferably is DNA.

The term “solid phase device” refers to a solid surface that allows themixing of a nucleic acid sample and DMA preferably in a liquid phase.Any container that provides suitable space for an interaction betweenthe nucleic acid sample and DMA would be suitable including silicon,glass, polymer film, plastics, or any other suitable surface. In variousembodiments the solid phase device includes a microfluidic device. Invarious other embodiments the solid phase device includes a ringresonator, most preferably the ring resonator is silicon based. Invarious embodiments the ring resonator comprises a waveguide structure.In various other embodiments other suitable solid phase devices known toa person skilled in the art could be used such as for example magneticbeads.

A “nucleic acid sample” may include any biological sample including:liquid blood or, liquid saliva, urine or blood, buccal swabs, hairs,bone, teeth, fingernails, tissues from any organs (including brain),muscle, skin, tumors, unknown lumps, biopsies including needle biopsies,cells or cell lines. The sample may be taken from any biologicalorganism, including plants, fungi and animal. The samples are preferablyfrom vertebrates including mammals and most preferably humans. Thesamples are preferably from cancerous tissue or tissues suspected ofbeing cancerous or forming neoplasm including breast cancer, lungcancer, pancreatic cancer, prostate cancer, bladder cancer, cervicalcancer, nasopharyngeal cancer, hepatocellular cancer, gastric cancer,colon cancer, stomach cancer, bone cancer, testicular cancer, thyroidcancer, lymphoma, leukemia, or any other known neoplasm. In variousembodiments samples may be taken from healthy non-cancerous tissue orbody fluids for comparison to cancerous tissue or body fluid samples. Invarious embodiments nucleic acid has been extracted from the sampleusing methods known in the art.

Dimethyl adipimidate (DMA) comprises a membrane permeable cross linkerthat contains amine reactive imidoester at each end of a 6 atom spacerarm. DMA is a non-chaotropic agent for directly binding nucleic acidincluding methylated DNA. The DMA comprises a structure of formula I:

In various embodiments the NH groups of DMA can be converted into theNH2+ groups with acids, with any suitable counter ion for example withhydrochloride salts or any other acids with a free Hydrogen group wouldbe suitable.

Without being limited to any theories it is postulated that and thebi-functional imidoester (amine reactive group) in DMA reacts with freeamino groups in DNA molecules, resulting in the cross-linking betweenDMA and DNA. The selective reaction between DMA and amine groups of thenucleic acid over that of protein may be related to DMA being positivelycharged therefore being attracted to negatively charged DNA compared tothe digested negatively charged protein fragments. Additionally, thefragmented DNA during the lysis step typically contains a few base pairsof single-stranded DNA at each end of the fragment, known as “sticky”ends, and DMA reacts with amine groups of “sticky” ends. Thebi-functional imidoester of DMA further provides high surface-area tovolume ratio for capturing DNA.

In various embodiments the method may further comprise functionalizingthe surface of the solid phase device to be aminated using surfacetreatments known in the art. The surface is functionalized prior toincubating the nucleic acid sample with the DMA. In various embodimentsthe surface is factionalized with an aminosilane preferably3-aminopropyltriethoxysilane (APTES). The DMA will interact with theaminated surface via covalent bonds. It is observed that the bondremains or is stronger when the DMA forms a complex with nucleic acid.The complex with DMA and DNA also reacts with the amine-modified surfaceand forms stable covalent bonds. The bonds are amidine bonds that arereversible at high pH (>pH 10) which can be used as a means to captureand release the complex by changing the pH.

In various embodiments the nucleic acid sample is extracted with aprotease prior to the incubation with the DMA, preferably proteinase K.The selective reaction between DMA and amine groups of the nucleic acidover that of protein may be related to the protease activity ofproteinase K. Proteinase K both lyses cells and digests most of theprotein eliminating the protein from the sample.

In various embodiments the nucleic acid comprises methylated DNA. Theterm “methylated DNA” has the normal meaning known in the art.Methylation of deoxyribonucleic acid (DNA) includes the addition of amethyl group on one or more cytosine or adenine at locations along anucleotide.

In various embodiments the solid phase device is a micro fluidic device.

The term “microfluidic device” includes microchips, microchannelstructures, or any suitable lab-on-a-chip platform known in the art. TheDMA technique can be applied to a solid phase based microfluidic deviceto isolate and purify nucleic acids as well as improve DNAamplification.

In various other embodiments the solid phase device is a ring resonator.

In various embodiments all steps for isolation and analysis of nucleicacid are performed on a solid phase device. The combination of low-costfabrication, high sensitivity and high multiplexing capability throughsmall dimensions makes microring resonator a good candidate fordisposable biosensor chips for point of care diagnostic test (POCT).

In various embodiments where the solid phase device is a ring resonatorthe method may further comprise

-   -   (i) determining an output light intensity measured by a detector        of an optical sensing system;    -   (ii) determining a change in an effective refractive index of a        resonator of the optical sensing system, during the incubation;

In various embodiments the method may further comprise eluting thecomplex. Eluting preferably refers to removing the complex of themethylated DNA with the DMA by disrupting any covalent bond formedbetween the complex and a surface of the solid phase device. A means ofremoving the complex of the nucleic acid with the DMA from the surfaceincludes using a high pH solution (>pH 10). One example high pH solutionfor removing the complex of the nucleic acid with the DMA from thesurface includes disrupting any covalent bond formed between the complexand the fictionalized surface would be sodium bicarbonate.

In various preferred embodiments the method may further comprisemodifying the complex at non-methylated cytosine residues. DNAmethylation analysis using bisulfite conversion can modifying thecomplex at non-methylated cytosine residues and can operate on pictogramquantities of the input DNA, the conversion leads to modification of atleast 50% of the input DNA, more preferably at least 60%, 70% or 80% ofthe input DNA, most preferably 90% of the input DNA.

In various embodiments the complex or the modified complex is amplifiedvia any method known in the art. DNA amplification can be carried out byvarious different methods, including PCR preferably with primersspecific for methylated and/or unmethylated DNA. The examples show thatthe efficiency of methylation specific PCR and genomic PCR amplificationare enhanced by using the method.

In various embodiments the method may further comprise detecting thecomplex or the modified complex. Detection may be made via any methodknown in the art including but not limited to sequencing methods,Southern blotting analysis or any other analysis technique known in theart.

Another aspect of the invention includes a system for isolating anucleic acid molecule of interest in a nucleic acid sample comprising:

-   -   (i) a Dimethyl adipimidate (DMA) compound capable of directly        binding the nucleic acid molecule; and    -   (ii) a solid surface for the interaction of the nucleic acid and        the DMA.

The nucleic acid sample, DMA and solid surface have the same meaning asdescribed earlier.

In various embodiments the solid surface is functionalized. Afunctionalized surface is a surface that has been aminated using surfacetreatments known in the art.

In various embodiments the surface is factionalized with an aminosilanepreferably 3-aminopropyltriethoxysilane (APTES).

In various embodiments the solid surface is on a micro fluidic device.In various other embodiments the solid surface is on a ring resonator.

In various embodiments the ring resonator is an optical detection sensorwherein said detection sensor has an altered reading when said nucleicacid molecule is bound to said DMA such that said sensor is configuredto sense a complex formed between the nucleic acid molecule and the DMA.

In various embodiments the optical detection sensor is configured toresonate at a resonant wavelength.

In various embodiments the system further comprises a tunable lasercapable of providing light at said resonant wavelength for the opticalsensor.

In various embodiments the optical sensor or ring resonator comprises awaveguide structure.

In various embodiments said resonator has a resonant wavelength thatshifts when said nucleic acid molecule is bound to said DMA, formingsaid complex.

In the following “Optical Sensing” will be described.

Detection of DNA methylation can be accomplished using an opticallybased system such as those known in the art. The system may include alight source, an optical sensor, and an optical detector. In variousembodiments, the light source outputs a range of wavelengths. Forexample, the light source may be a relatively narrow-band light sourcethat outputs light having a narrow bandwidth wherein the wavelength ofthe light source is swept over a region many times the bandwidth of thelight source. In various embodiments the range of wavelengths may be(400-700 nm (visible), 700-400 (IR-A), 1260-1360 nm (O band), 1360-1460nm (E band), 1460-1530 nm (S band), 1530-1565 nm (C band), and 1565-1625nm (L band). This light source may, for example, be a laser. This lasermay be a tunable laser such that the wavelength of the laser output isvaried. In some embodiments the laser is a diode laser having anexternal cavity. This laser need not be limited to any particular kindand may, for example, be a fiber laser, a solid state laser, asemiconductor laser or other type of laser or laser system. The laseritself may have a wavelength that is adjustable and that can be scannedor swept. Alternatively, additional optical components can be used toprovide different wavelengths. In some embodiments, the light sourceoutput light having a wavelength for which the waveguide structure issufficiently optically transmissive. In some embodiments, the waveguidestructure is within a sample medium such as an aqueous medium and thelight source outputs light having a wavelength for which the medium issubstantially optically transmissive such that resonance can be reachedin the optical resonator. Additionally, in some embodiments, the lightsource output has a wavelength in a range where the complex does nothave a non-linear refractive index. Likewise, in various embodiments,the light source may be a coherent light source and output light havinga relatively long coherence length. However, in various embodiments, thelight source may be a coherent light source that outputs light having ashort coherence length. For example, in certain embodiments, a broadbandlight source such as a super-luminescent light emitting diode (SLED) maybe used. In such cases, the wavelength need not be swept.

The light source provides light to the optical sensor. The light sourcemay be controlled by control electronics. These electronics may, forexample, control the wavelength of the light source, and in particular,cause the light source to sweep the wavelength of the optical outputthereof. In some embodiments, a portion of the light emitted from thelight source is sampled to determine, for example, the emissionwavelength of the light source.

In some embodiments, the optical sensor comprises a transducer thatalters the optical input based on the presence and/or concentration ofthe complex to be detected. The optical sensor may be a waveguidestructure. The optical sensor may be an integrated optical device andmay be included on a chip. The optical sensor may comprise semiconductormaterial such as silicon. The optical sensor may be an interferometricstructure (e.g., an interferometer) and produce an output signal as aresult of optical interference. The optical sensor may be included in anarray of optical sensors.

The optical detector detects the optical output of the sensor. Invarious embodiments, the optical detector comprises a transducer thatconverts an optical input into an electrical output. This electricaloutput may be processed by processing electronics to analyze the outputof the sensor. The optical detector may comprise a photodiode detector.Other types of detectors may be employed. Collection optics in anoptical path between the sensor and the detector may facilitatecollection of the optical output of the sensor and direct this output tothe detector. Additional optics such as mirrors, beam-splitters, orother components may also be included in the optical path from thesensor to the detector.

In various embodiments, the optical sensor is disposed on a chip whilethe light source and/or the optical detector are separate from the chip.The light source and optical detector may, for example, be part of anapparatus comprising free space optics that interrogates the opticalsensors on the chip, as will be discussed in more detail below.

In various embodiments, a solution such as DNA sample solution is flowedpast the optical sensor. The detector detects modulation in an opticalsignal from the optical sensor when a complex formed between the nucleicacid and the DMA is detected.

Ring resonators offer highly sensitive optical sensors that can beprepared so as to detect a complex formed between the nucleic acid andthe DMA. The operation of a ring resonator may comprise any suitableconfiguration. In various embodiments the optical sensor comprises aninput/output waveguide having an input and an output and a ringresonator disposed in proximity to a portion of the input/outputwaveguide that is arranged between the input and the output. The closeproximity facilitates optical coupling between the input/outputwaveguide and the ring resonator, which is also a waveguide. In thisexample, the input/output waveguide is linear and the ring resonator iscircular such that light propagating in the input/output waveguide fromthe input to the output is coupled into the ring resonator andcirculates therein. Other shapes for the input/output waveguide and ringresonator are also possible.

In various embodiments, the light injected into the waveguide inputincludes a range of wavelengths, for example, from a narrow band lightsource having a narrow band peak that is swept over time (or from abroadband light source such as a super-luminescent diode). Similarly, anoutput spectrum takes the form of a waveguide output. A portion of thisoutput spectrum may be expanded into a plot of intensity versuswavelength in the spectral distribution at the resonance wavelength, ofthe ring resonator.

Other configurations are possible, for example, other layers may beadded (or removed) or patterned differently. A portion of the substratemay have a linear waveguide and ring resonator formed thereon may bepart of a larger integrated optical chip.

As is well known, light propagates within waveguides via total internalreflection. The waveguide supports modes that yield a spatially varyingintensity pattern across the waveguide. A portion of the electric fieldand optical energy referred to as the evanescent “tail” lies outside thebounds of the waveguide. An object located close to the waveguide, forexample, within this evanescent field length affects the waveguide. Inparticular, objects within this close proximity to the waveguide affectthe index of refraction of the waveguide. The index of refraction, n,can thus be different when such an object is closely adhered to thewaveguide or not. In various embodiments, for example, the presence ofan object increases the refractive index of the waveguide. In thismanner, the optical sensor may be perturbed by the presence of an objectin the vicinity of the waveguide structure thereby enabling detection.In various embodiments, the size of the particle is about the length ofthe evanescent field to enhance interaction there between.

In the case of the ring resonator, an increase in the refractive index,n, increases the optical path length traveled by light circulating aboutthe ring. Longer wavelengths can resonate in the resonator and, hence,the resonance frequency is shifted to a lower frequency. The shift inthe resonant wavelengths of the resonator can therefore be monitored todetermine if an object has located itself within close proximity to theoptical sensor (e.g., the ring resonator and/or a region of the linearwaveguide closest to the ring resonator). A binding event, whereby acomplex formed between the nucleic acid and the DMA, can thus bedetected by obtaining the spectral output from the waveguide output andidentifying dips in intensity (or peaks in attenuation) therein and theshift of these dips in intensity.

In various embodiments, the waveguide and/or the ring resonator comprisesilicon. In some embodiments, the surface of the waveguide may benatively passivated with silicon dioxide. As a result, standard siloxanechemistry may be an effective method for introducing various reactivemoieties to the waveguide, which are then subsequently used tocovalently fix the complex via a range of standard bioconjugatereactions.

Moreover, the linear waveguide, ring resonator, and/or additionalon-chip optics may be easily fabricated on relatively cheapsilicon-on-insulator (SOI) wafers using well established semiconductorfabrication methods, which are extremely scalable, cost effective, andhighly reproducible. Additionally, these devices may be easilyfabricated and complications due to vibration are reduced when comparedto “freestanding” cavities. In one example embodiment, 8″ SOI wafers mayeach contain about 40,000 individually addressable ring resonators. Oneadvantage of using silicon-based technology is that various embodimentsmay operate in the Si transparency window of around 1.55 μm, a commonoptical telecommunications wavelength, meaning that lasers and detectorsare readily available in the commercial marketplace as plug-and-playcomponents.

In various embodiments the system comprises a silicon microringresonator for the label-free analysis and isolation of nucleic acid suchas methylated DNA from genomic DNA from cancer samples on solid phasedevice. DNA methylation analysis is of great importance for cancerresearch and clinics, since it enables earlier cancer diagnosis prior tothe point of metastasis. In order to analyze the DNA methylation, theisolation and analysis of the methylated DNA from whole genomic DNA byusing high sensitivity and specificity methods are the most importantfactor for detection of DNA methylation. Recent reports suggest highlysensitive silicon-based microring resonators can be used to detectbiomolecules (e.g., protein, methylated DNA, nucleic acids) bymonitoring a shift in the resonant wavelength.

In various embodiments, light may be directed into an input of the firstinput/output waveguide, and, depending on the state of the first ringresonator and the wavelength of light, may be directed to either anoutput of the first waveguide, or may be directed into a secondwaveguide. For example, for the resonant wavelengths of the first ringresonator, the light may be coupled into the second waveguide instead ofbeing output from the first waveguide at output. Light coupled into thesecond waveguide from the first ring resonator may be directed to eitheran output of the second waveguide or into the third waveguide, dependingon the state of the third ring resonator. For example, for the resonantwavelengths of the third ring resonator, the light may be coupled intothe third waveguide and then output at an output location. In the casewhere the light source that directs light into the first input/outputwaveguide comprises a broadband light source such as a super-luminescentdiode that outputs a broadband spectrum, the light referred to above maybe a wavelength component of the broader spectrum.

Other configurations can be used. A tunable laser or other tunable lightsource may be used as the input source and the wavelength of the outputof the tunable laser can be swept. Alternatively, a broadband lightsource such as a super luminescent diode may be used.

More ring resonators may be added. Additionally, the ring resonators maybe positioned differently with respect to each other as well as withrespect to the input/output waveguide.

Various embodiments of ring resonators and possibly other geometriesrepeatedly circulate light around, for example, their perimeter,dramatically increasing the optical path length. Furthermore,interference between photons circulating in the structure and thosetraversing the adjacent waveguide create a resonant cavity ofextraordinarily narrow spectral line width resulting in a high-Q device.The resulting resonance wavelengths are quite sensitive to changes inthe local refractive index. As discussed herein, this sensitivityenables the sensors to detect small masses.

Therefore, the proposed concept could be very useful for DNA methylationanalysis in cancer research and clinic applications.

EXAMPLES

Silicon microring resonator chips 200 mm SOI wafer with 220 nm thick topsilicon layer and 2 μm think buried oxide layers by 248 nm deep UVlithography were purchased commercially then, wave-guides, and gratingswere patterned thereon etched to buried oxide layer by reactive ionetching (RIE) process, followed by the deposition of 1.5 μm PECVD SiO2as a top cladding layer. An array of microrings consisted of four ringsthat were connected to one common input waveguide (through) and eachring had a dedicated output waveguide (drop). Three of the four ringswere used as sensor rings where windows were opened over selectedindividual sensor ring via the combination of dry and wet etching. Oneof the rings was used as a reference sensor to monitor temperatureinduced drift. The rings are race-track style rings with a radius of 5μm, coupling length varied between 2 and 2.042 μm to avoid spectraloverlap of resonances. The output signals of the three rings werecollected via a vertical grating coupler to single-mode fiber opticprobe. Insertion loss (IL) spectrum was measured with EXFO IQS-12004BDWDM passive component test system.

First, DMA (dimethyl adipimidate) was used as a chemical agent forcapturing of the methylated DNA on silicon microring resonator, whichwill be monitored and analyzed the methylated DNA binding in real-timemanner. The DMA has been described as a new antisickling agent by use ofthe bifunctional cross-linking reagents, which is known to linkcovalently the free amino groups in polypeptides. After capturing of themethylated DNA with the DMA, the methylated DNA is eluted by sodiumbicarbonate (pH 10.6). Then, methylation specific PCR after bisulfitemodification is performed to verify the efficiency for the isolation ofthe methylated DNA (FIG. 1, Black_round dot). The protocol was modifiedfrom those described previously. Briefly (FIG. 2, #1), the device wasfirst treated with oxygen plasma. Then it was immersed in a solution of2% 3-aminopropyltriethoxysilane (APTES) in a mixture of ethanol/H₂O(95%/15%, v/v) for 2 hours followed by thoroughly rinsing it withethanol and de-lionized water. It was then dried under a nitrogen streamand heated at 120° C. for 15 minutes to cure the chips.

Genomic DNA was extracted from cancer cell lines (T24 and MCF7) usingproteinase K and QIAamp DNA Mini Kit (Hilden, Germany). The T24 cellline, an epithelial line derived from human urinary bladder transitionalcells, and the MCF7 cell line, an epithelial line derived from humanmammary gland cells, were used for extraction of genomic DNA. The cancercell lines were purchased from ATCC (American type culture collection,Manassas, Va.).

The sensor chips were then incubated with mixture of 1 ug of the genomicDNA and DMA (10 mg/ml) in PBS for 30 min. The wavelength shift wascollected every 5 min up to 30 min after hybridization. After thebinding, excess DNA target was rinsed free from the surface by washingthe chip two times for 10 minutes each with PBS buffer and measured.Finally, the methylated DNA remained on the surface and then themethylated DNA was eluted by sodium bicarbonate (pH 10.6).

Alternatively, for the comparison the MBD protein was used for capturingof the methylated DNA on solid phase (FIG. 2, #2), not in solutionphase. Briefly, the sensor surface was first functionalized with APTES,as described in above. The sensor chip was then incubated with 1 mg/mLNHS-biotin in de-ionized water for 1 h and rinsed with de-ionized water.The binding assay between biotin and streptavidin was performed byapplying streptavidin solution in PBS (190 pM-950 nM). The sensor chipswere then incubated with mixture of 1 μg of the genomic DNA andBiotin-MBD protein mixture in PBS for 30 min. The wavelength shift wascollected every 5 min up to 30 min after hybridization. After thebinding, excess DNA target was rinsed free from the surface by washingthe chip two times for 10 minutes each with PBS buffer and measured.Finally, the methylated DNA remained on the surface and then themethylated DNA was eluted by sodium bicarbonate (pH 10.6). Allexperiments were carried out at room temperature.

FIG. 3 shows experimental results for the method. Extracted genomic DNAfrom cancer cell lines such as T24 (Bladder) and MCF7 (Breast) was usedfor the isolation of the methylated DNA by using the solid phase device(FIG. 4B). The wavelength shift was highly observed when the sample wasadded and the mixture of the methylated DNA and DMA formed a complex onthe functionalized solid phase surface (FIG. 3A). Then, the shift waspartially reduced by washing with PBS due to getting rid of excess DNA.

The isolated DNA bound to the surface by covalent bond after washing.Finally, the DNA was collected by sodium bicarbonate for DNA analysis.The eluted DNA from T24 extracted genomic DNA was used for genetic andepigenetic analysis via conventional PCR and methylation-specific(MS)-PCR. In order to verify the efficiency of the method, Methylationspecific PCR was performed for detection of the DNA methylation of theRARβ gene by using the isolated methylated DNA. The results showed thatthe PCR band is stronger in method (#1) than the conventional method(FIG. 3B).

In summary, new methods for isolation and analysis of the methylated DNAon solid phase device including silicon microring resonators isdescribed. DMA is a chemical agent for capturing the methylated DNA withhigh specificity on solid phase device. The methods can provide not onlyhigh efficiency of the methylated DNA isolation, but also monitoring theanalysis of the methylated DNA in label free and real-time manner. Themethod could be very useful for DNA methylation analysis in cancerresearch and clinic applications.

To elucidate the effect of DMA as a solid-phase-based extractionreagent, PCR-based DNA amplification was performed by using the purifiedor extracted DNA via the DMA method. All primers used for conventional,MS-PCR and real-time PCR of the genes (RARβ, HRAS, and Actin) are knownin the art. Conventional PCR and MS-PCR were performed to verify theefficiency of the proposed technique for the genetic and epigeneticanalysis. For bisulfite conversion of DNA prior to the MS-PCR operation,we used either 50 μl of purified DNA by the proposed technique or 1 μgof the extracted genomic DNA from the T24 cell line (ATCC), anepithelial cell line derived from human urinary bladder transitionalcells, and the CpGenome DNA modification kit (Millipore, Billerica,Mass.). The T24 human bladder cancer cell line was maintained in plasticculture dishes with high glucose Dulbecco's modified Eagle's Medium(DMEM, Life Technology) supplemented with 10% fetal calf serum (FCS) ina 37° C. humid incubator with 5% ambient CO₂. The cancer cell lines werecultured, and then the genomic DNA was extracted by using AL buffer withproteinase K from a QIAmp DNA mini kit (Qiagen, Hilden, Germany).Briefly, for genetic analysis of HRAS gene, 2 μl of the eluted DNA fromeach sample such as the complex, DMA alone, and DNA alone was amplifiedin a total volume of 25 μl containing 1×PCR buffer (Qiagen, Hilden,Germany), 2.5 mM MgCl₂, 0.25 mM deoxynucleotide triphosphate, 25 pmol ofeach primer (for HRAS gene), and 1 unit of Taq DNA polymerase (Qiagen,Hilden, Germany) at 95° C. for 15 min; 35 cycles of 95° C. for 30 s, 60°C. for 30 s, and 72° C. for 30 s; and a final elongation step at 72° C.for 7 min. For the epigenetic analysis of RARβ gene, 2 μl ofbisulfitemodified DNA from either conventional (no purification step) orthe proposed DMA method was amplified in a total volume of 25 μlcontaining 1×PCR buffer (Qiagen, Hilden, Germany), 2.5 mM MgCl2, 0.25 mMdeoxynucleotide triphosphate, 25 pmol of each primer (for RARβ gene),and 1 unit of Taq DNA polymerase (Qiagen, Hilden, Germany) at 95° C. for15 min; 45 cycles of 95° C. for 30 s, 59° C. for 30 s, and 72° C. for 30s; and a final elongation step at 72° C. for 7 min. PCR amplicons werevisualized by gel electrophoresis, which was used to separate PCRproducts on a 2% agarose gel containing ethidium bromide (EtBr)(Sigma-Aldrich). The gel was visualized using a Gel Doc System(Bio-Rad). The band intensity was calculated by Image J (NationalInstitute of Health, USA). Determination of DNA concentration and puritywas done by UV spectrophotometer. Real-time PCR was performed to verifythe efficiency of the 3 different extraction methods.

The target template for RT-PCR was obtained from human genomic DNAextracted from either whole blood or urine samples. For real-time PCR,the following procedure is modified from the protocol supplied withLightCycler 2.0 (Roche Diagnostics). Briefly, 5 μl of DNA was amplifiedin a total volume of 20 μl, containing 4 μl of LightCycler FastStart DNAMaster mix, 25 pmol of each primer, and 2 μl of DNA template. An initialpre-incubation cycle of 95° C. for 10 min was followed by 45 cycles at95° C. for 10 s, and 60° C. for 30 s (for HRAS and Actin genes); and bya cooling step at 40° C. for 30 s. The amplified products with SYBRGreen signals were carried out on a LightCycler 2.0.

The DMA technique in microfluidic chips was compared with 2 differentDNA extraction methods (QIAmp DNA mini kit in solution phase and achaotropic reagent (ethanol) on a micro-chip) to validate the efficiencyof the DMA reagent in SPE of DNA from human whole blood and urinesamples in micro-chips (FIG. 4A). First, either 2 μl of whole blood(with 8 μl of PBS) or 10 μl of urine (without pH adjustment) were usedfor DNA extraction in microfluidic chips. For obtaining the extractedDNA, the process consisting of five steps was followed: (1) filteringand separating the cells based on their size, (2) lysing the cells, (3)binding the DNA with either a chaotropic reagent or DMA, (4) washing andpurifying the DNA, and (5) eluting the DNA with either distilled wateror elution buffer. After elution of the extracted DNA, the purity of theDNA extracted by the 3 different techniques was measured determining theratio of the optical densities of the samples at 260 nm (DNA) and 280 nm(protein). The quality of the DNA from the DMA technique in both bloodand urine was statistically significant induced purity for the sample(p<0.001) compared to that obtained from the ethanol based chaotropicmethod. In fact, since the urine medium is relatively clean and containsfew proteins compared to blood, it was expected that the effectivenessof the DMA-based SPE on the quality of DNA extracted from the bloodsample over other methods would be more pronounced than that from theurine sample. In contrast to expectation, highly purified DNA by theproposed technique was obtained from both the whole blood and urinesamples. It is assumed that this is because the DMA tightly binds withDNA molecules extracted from the fluids samples during the washing stepto get rid of other molecules such as cell debris, proteins, and so on,compared to the chaotropic method. Next, the amplification test wasperformed with the extracted DNA using RT-PCR. Both HRAS and Actin geneswere used as genetic targets and amplified in all DNA samples extractedby using the 3 different techniques. It was shown that the quantity andquality of the DNA extracted with the DMA-based technique was greaterthan that obtained by the other methods. For real-time (RT)-PCR, 2 μl ofDNA extract from total DNA sample groups was used without anyquantification in order to mimic the real situation for the micro totalanalysis system in which the extracted DNA from the device is morelikely directly used as a target template without any quantification forgenetic analysis in clinical settings. Thus, the SYBR fluorescencesignal in use of the QIAmp DNA mini kit (DNA extracted from 200 μl ofwhole blood or urine) appeared to be saturated at an earlier Ct (cyclethreshold) value than others. It was observed that the DNA biomarkers(HRAS, Actin) for the genetic analysis were amplified with good qualityand quantity by using the DMA-based technique. Therefore, the proposedDMA technique in this study can be useful for the solid phase DNAextraction with high quality from a small volume of human body fluids ina micro-chip system.

Design and Fabrication of Silicon Microfluidic Devices

For testing DMA-based method in a microchip environment, silicon basedDNA extraction microfluidic devices was used. The structure andfabrication of the DNA extraction microfluidic devices are known. Thesilicon microfluidic chip comprises three components, including (1)pre-filtration part for cell separation; (2) micromixer consisting of atwo-stage spiral mixer for cell lysis; and (3) a meander-shapedmicrochannel for DMA-based method for maximization of the SiO₂ surfacearea, which is estimated to be over 60 mm2. The microfluidic chips werefabricated using a reactive ion etching (RIE) process on the front sideof the silicon substrate overlaid with 2 μm-thick thermal SiO₂ as a hardmasking layer. To form the fluidic interconnections, a wet etch processwith potassium hydroxide (KOH) was applied to the back side of thesilicon substrate overlaid with a composite masking layer of siliconnitride (Si3N4) deposited by low pressure chemical vapor deposition(LPCVD) over a thermal SiO₂ layer. With the remaining SiO₂ thin filmlayer on the micro channel surface, an anodic bonding process with aPyrex glass substrate was performed to cap the open channel. The overallsize of each fabricated microfluidic device is 16 mm×12 mm×1.2 mm.

The microfluidic device process flow for DNA extraction from human bodyfluids (whole blood or urine) with a small sample volume includes thefollowing steps that were modified from previously reportedprotocol.24-35 The microfluidic chips are packaged in a polycarbonatehousing, which includes o-rings with drilled holes for fluidicinterconnection from external syringe pumps to the bottom of the siliconmicro-chip. All samples and reagents are sequentially delivered to themicrochip in the following order Inlet I: separation—injecting thesamples into the microfilter to separate cells by size; Inlet II:secretion—injecting the AL buffer with proteinase K to be used as alysis buffer into the micromixer Inlet III: washing andelution—injecting the wash buffers (either EtOH or DMA and PBS) topurify the sample; and Outlet IV: eluting the nucleic acid from siliconsolid surface. When using a chaotropic method (ethanol), either thewhole blood (2 μl) with 8 μl of PBS buffer or urine (10 μl) wereinjected with a syringe pump (KD Scientific. MA) into Inlet I at a flowrate of 1.67 μl min-1 for 10 min. Then, lysis buffers were injected intoInlet II at a flow rate of 3 μl min-1 for 10 min, with an increase inInlet I flow rate to 3 μl min-1 for 10 min with PBS buffer by syringepumps. Ethanol was added to Inlet III at a flow rate of 10 μl min-1 for10 min during the lysis reaction. Then, ethanol alone was sequentiallyused for washing through the chip at 12.5 μl min-1 for 5 min. Finally,the extracted DNA was eluted with pure water at 12.5 μl min-1 for 10min.

When using the proposed DMA method, the microchannel (Inlet III) partwas first coated with APTES for 2 h with a syringe pump, and the surfacewas washed with ethanol for 20 min. Then the surface was dried withnitrogen gas to ready for reaction. Either the whole blood (2 μl) with 8μl of PBS buffer or urine (10 μl) was injected with a syringe pump intoInlet I at a flow rate of 1.67 μl min-1 for 10 min. Lysis buffers werethen injected into Inlet II at a flow rate of 3 μl min-1 for 10 min, andthe Inlet I flow rate was increased to 3 μl min-1 for 10 min with PBSbuffer by syringe pumps. DMA solution (25 mg mL-1) was added to InletIII at a flow rate of 10 μl min-1 for 10 min during the lysis reaction.Then, the PBS buffer was sequentially passed through the chip, at 12.5μl min-1 for 10 min, in order to get rid of non-specific bound moleculesand PCR inhibitors. Finally, the extracted DNA was eluted with elutionbuffer at 12.5 μl min-1 for 10 min. In addition, 200 μl of whole bloodor urine was used for genomic DNA extraction as reference material usinga QIAmp DNA mini kit (Hilden, Germany). All extracted DNA was then usedfor genetic analysis by RT-PCR.

By “comprising” it is meant including, but not limited to, whateverfollows the word “comprising”. Thus, use of the term “comprising”indicates that the listed elements are required or mandatory, but thatother elements are optional and may or may not be present.

By “consisting of” is meant including, and limited to, whatever followsthe phrase “consisting of”. Thus, the phrase “consisting of” indicatesthat the listed elements are required or mandatory, and that no otherelements may be present.

The inventions illustratively described herein may suitably be practicedin the absence of any element or elements, limitation or limitations,not specifically disclosed herein. Thus, for example, the terms“comprising”, “including”, “containing”, etc. shall be read expansivelyand without limitation. Additionally, the terms and expressions employedherein have been used as terms of description and not of limitation, andthere is no intention in the use of such terms and expressions ofexcluding any equivalents of the features shown and described orportions thereof, but it is recognized that various modifications arepossible within the scope of the invention claimed. Thus, it should beunderstood that although the present invention has been specificallydisclosed by preferred embodiments and optional features, modificationand variation of the inventions embodied therein herein disclosed may beresorted to by those skilled in the art, and that such modifications andvariations are considered to be within the scope of this invention.

By “about” in relation to a given numerical value, such as fortemperature and period of time, it is meant to include numerical valueswithin 10% of the specified value.

The invention has been described broadly and generically herein. Each ofthe narrower species and sub-generic groupings falling within thegeneric disclosure also form part of the invention. This includes thegeneric description of the invention with a proviso or negativelimitation removing any subject matter from the genus, regardless ofwhether or not the excised material is specifically recited herein.

Other embodiments are within the following claims and non-limitingexamples. In addition, where features or aspects of the invention aredescribed in terms of Markush groups, those skilled in the art willrecognize that the invention is also thereby described in terms of anyindividual member or subgroup of members of the Markush group.

What is claimed is:
 1. A method of isolating a nucleic acid molecule ona solid phase device, the method comprising: incubating a nucleic acidsample with dimethyl adipimidate (DMA) under conditions which allow theDMA to bind directly to the nucleic acid molecule to form a complex ofthe nucleic acid molecule with the DMA; contacting the complex with asurface of the solid phase device; and isolating the complex from thesurface using an elution solution having a pH of more than
 10. 2. Themethod according to claim 1, wherein a surface of the solid phase deviceis functionalizing prior to the incubation.
 3. The method according toclaim 1, wherein the nucleic acid sample is extracted with a protease.4. The method according to claim 3, wherein the protease is proteinaseK.
 5. The method according to claim 1, wherein the nucleic acid moleculecomprises methylated DNA.
 6. The method according to claim 1, whereinthe solid phase device is a microfluidic device.
 7. The method accordingto claim 1, wherein the solid phase device is a ring resonator.
 8. Themethod according to claim 7, further comprising determining an outputlight intensity measured by a detector of an optical sensing system;determining a change in an effective refractive index of a resonator ofthe optical sensing system, during the incubation.
 9. The methodaccording to claim 7, wherein said ring resonator comprises a waveguidestructure.
 10. The method according to claim 1 wherein the complex ismodified at non-methylated cytosine residues.
 11. The method accordingto claim 1 wherein the complex or the modified complex is amplified. 12.The method according to claim 1 wherein the complex or the modifiedcomplex is detected.
 13. The method according to claim 1, furthercomprising: analyzing the nucleic acid molecule of the complex.